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For the first time, the scientists determined the three dimensional structure of a protein by the method of femtosecond nanocrystallography, a highly innovative technique that was developed by the team at ASU and their collaborators using X-ray laser diffraction from the LCLS free-electron laser.
Contradictory to popular opinion, this technique successfully demonstrates that high quality data can be acquired so quickly that reaction chemistry involving proteins can now be studied in real time. This method goes well beyond form, but goes into function where changes in a molecule can be seen in action.
“The ASU team is one of the pioneers of the method of fs crystallography," said ASU Regents' Professor John Spence. "Our work includes the design of a sample delivery system, the identification and discovery of nanocrystals, as well as the development of the theory and computer algorithms used to analyze the data. The method is so exciting as its further development will allow us to determine movies of molecular machines at work."
The technique – described in the paper “High-resolution protein structure determination by serial femtosecond crystallography” – works at a higher resolution than previously achieved using X-ray lasers, allowing scientists to use smaller crystals than typical with other methods that grow inside living cells, and could enable researchers to view molecular dynamics at a time-scale never observed before. SLAC’s Linac Coherent LightSource (LCLS) shines a billion times brighter than traditional synchrotron X-ray sources.
Science stated that “the grand goal is to push X-ray diffraction to its ultimate limit and use an X-ray laser to decipher a protein structure by zapping individual molecules.” According to Science, the success of the study "shows the potential of X-ray lasers to decipher proteins that conventional X-ray sources cannot.”
Advancements in this research may be of great importance for the development of new drugs to fight African sleeping sickness, which kills approximately 30,000 people each year. This method shows novel features of the structure of the CatB protein, a protease that is essential for the pathogenesis, including the structure of the natural inhibitor peptide bound in the catalytic cleft of the enzyme. The discovery of the enzyme’s 3D structure has enabled the researchers to pinpoint distinctive structural differences between the human and the parasite’s form of the enzyme.
The research is accomplished by a large international team which involves, in addition to ASU, key institutions including the Center of Free-Electron Laser Science at DESY in Hamburg, University of Luebeck, who grew the crystals of CatB and the Max Plank Institute in Heidelberg. Henry Chapman from the Center of Free-Electron Laser Science led the team of scientists for this study.
ASU contributors include the research groups of Spence, Bruce Doak and Uwe Weierstall from the Department of Physics, and Galvin Professor Petra Fromme from the Department of Chemistry and Biochemistry, all part of the College of Liberal Arts and Sciences.
“In 2009 we showed the first proof of principle after the world’s first high-energy free-electron laser had become operational in Stanford,” said Fromme. “This leading technology will revolutionize the field of structural biology.”
“This research would not have been possible without the liquid jet injector for the nanocrystals developed at ASU (patent has been filed) nor the biochemical expertise that led to success in the preceding measurements, which laid the groundwork for the Trypanosoma work,” said Doak, professor from ASU’s Department of Physics.
The project depends on the excellent team of ASU’s research scientists, postdoctoral researchers and graduate students from Physics and Chemistry who work at ASU and travel with their professors to conduct the experiments at Stanford at the LCLS free-electron laser. These include faculty research associates Raimund Fromme, Ingo Grotjohann and Tzu-Chiao Chao; Nadia Zatsepin, post-doctoral researcher, graduate students Christopher Kupitz (Biochemistry) and Dingjie Wang (Physics); as well as Mark Hunter and Richard Kirian who graduated with PhDs from ASU in Chemistry and Physics respectively and now work on the femtosecond crystallography project at Lawrence Livermore National Laboratory and DESY (Deutsches Elektronen Synchrotron).
“ASU is proud of the achievements and dedication of these scientists for their innovative work in producing high-quality and groundbreaking discoveries that advance use-inspired research to combat disease,” said William Petuskey, associate vice president of natural/physical sciences and engineering/technology for ASU’s Office of Knowledge Enterprise Development. “This breakthrough will help pave the way for further advancements in biomedical research.”
The ASU team was awarded a patent for the sample delivery system, which uses microscopic (and even nanoscopic) liquid jets to inject samples into the X-ray beam.